Download Sourcing human embryos for embryonic stem cell lines

Review Article
Indian J Med Res 140 (Supplement), November 2014, pp 106-111
Sourcing human embryos for embryonic stem cell lines:
Problems & perspectives
Rajvi H. Mehta
Trivector Embryology Support Academy, Mumbai, India
Received April 12, 2013
The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos
following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique.
These cell lines could have been successfully used to increase our understanding of human developmental
biology, transplantation medicine and the emerging science of regenerative medicine. The main source
for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It
is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive
technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these,
only two or maximum of three embryos are transferred while the rest are cryopreserved as per the
decision of the couple. in case a couple does not desire to ‘cryopreserve’ their embryos then all the
embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in
need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised
by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos?
The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the
embryos is primarily assessed based on morphology and the rate of development mainly judged by single
point assessment. Despite many criteria described in the literature, the quality assessment is purely
subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria
for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos?
This paper discusses some of the newer methods to procure embryos for the derivation of embryonic
stem cell lines which will respect the ethical concerns but still provide the source material.
Key words cryopreserved - embryonic stem cell lines - hESC - human embryos
Introduction
to differentiate into any of the three embryonic germ
layers, including gut epithelium (endoderm); cartilage,
bone, smooth muscle, and striated muscle (mesoderm);
and neural epithelium, embryonic ganglia, and stratified
squamous epithelium (ectoderm). Thomson et al1 were
the first to report on the successful derivation of human
embryonic stem cell (hESC) lines. They prophesised in
Embryonic stem cell lines are derived from cells
of the early mammalian embryos. The two main
characteristics of these cell lines are their capability
of unlimited and undifferentiated proliferation in vitro.
Even after four to five months of undifferentiated
proliferation in vitro, these cells maintain the potential
106
Mehta: Sourcing human embryos for embryonic stem cell lines
their path-breaking report on the potential application
of this technique1, “Many diseases, such as Parkinson’s
disease and juvenile-onset diabetes mellitus, result
from the death or dysfunction of just one or a few
cell types. The replacement of those cells could offer
lifelong treatment”1.
Potential applications and significance of human
embryonic stem cells (hESCs)
The potential application of human ESC lines has
generated tremendous interest not only in the scientific
fraternity but also the general public. These cell lines
provide new tools for the study of molecular mechanisms
involved in normal human development, which cannot,
for obvious ethical reasons, be studied in utero. This
will aid our understanding of the formation of normal
tissues from the embryonic stage to comprehend
the development of organs and also to explore the
oncogenesis and development of tumours of embryonic
origin. In theory, by their capacity for self-renewal,
hESCs can generate an unlimited number of specialized
differentiated cells, providing a very interesting source
of cells for cell therapy and thereby opening the
doors to the science of regenerative medicine. One
of the greatest potentials of the ESCs would be their
ability to differentiate into cardiomyocytes among the
other cell types. Cardiomyocytes become terminallydifferentiated soon after birth and lose their ability to
proliferate and there is no evidence that stem/progenitor
cells derived from other sources, such as the bone
marrow or the cord blood, are able to give rise to the
contractile heart muscle cells following transplantation
into the heart. The genetically stable human embryonic
stem cells (hESCs) with their unlimited expansion
ability and unrestricted plasticity, proffer a pluripotent
reservoir for in vitro derivation of large supplies of
human somatic cells that are restricted to the lineage in
need of repair and regeneration2.
To achieve this potential application of hESC, the
Department of Biotechnology (DBT) and the Indian
Council of Medical Research (ICMR) (2007) approved
research for derivation of hESC lines for specific
reasons3: (i) Develop methods to detect abnormalities in
embryos before implantation. (ii) Advance knowledge,
which can be used for infertility treatment or improving
contraception techniques. (iii) Increase knowledge
about causation of serious diseases and their treatment
including tissue therapies. (iv) Developing methods of
therapy for diseased or damaged tissues or organs.
107
The limiting factors
It had been anticipated that it would be a long
while before ESC would find clinical application. This
was due to the current inadequate understanding on
how to induce differentiation of these cell lines into
the desired cell types and potential risk of tumour
formation. Another important challenge for the clinical
use of these cells is the issue of immunocompatibility
which can lead to the rejection of these cells following
hES cell transplantation. Such a problem may be dealt
with by the establishment of hES cell banks to attend
different populations. The ESC lines can be derived
from only 8-13 per cent of the blastocysts4 and the
rate of formation of blastocysts in vitro necessitates a
large number of embryos. The source and availability
of human embryos for the derivation of hESC become
a challenge in itself because of the immense ethical
implications.
Ethical concerns with the use of hESC lines
The sourcing of human embryos for the derivation
of human ESC line has been routed in controversy.
hESC research has been shrouded by fears about human
cloning, the commodification of human biological
material, the mixing of human and animal species,
and the hubristic quest for regenerative immortality5.
Opponents of hES cell research have maintained that
all preimplantation embryos have the potential to
become full-fledged human beings and that it is always
morally wrong to destroy this potential. All the ethical
debates are pertaining to the moral status of the embryo
and thereby intractable restricting research.
Sourcing of embryos for derivation of hESCs
The primary source of human embryos is the assisted
reproductive technology (ART) programmes. This
section reviews the common current sources of human
embryos for the derivation of human ESC and their
limitations and the some novel ideas being explored.
Good quality spare embryos: It is a common practice
in ART programmess to subject the woman to ovarian
stimulation so as to retrieve multiple oocytes. These
oocytes following in vitro fertilization (IVF) lead
to multiple embryos, of which a maximum of three
embryos are transferred. ‘Spare’ embryos are obtained
in most ART cycles. The good quality ones are
cryopreserved for future use while the poor quality
embryo are discarded. As treatment of infertility is
the reason for couples to undergo ART most couples
would not be comfortable with the idea of ‘giving
108 INDIAN J MED RES, november (Suppl.) 2014
away’ their good embryos till their primary goal of
achieving pregnancy is attained. A few couples do not
prefer to cryopreserve embryos because of ethical or
financial reasons and such embryos could be donated
for research on ESC lines.
The questions and concerns that could be raised
against the use of such ‘spare’ embryos are: (i) Could
a woman be ‘intentionally’ over-stimulated so as to
ensure a good number of ‘spare’ embryos?, and (ii)
Would it be fair to subject embryos for research from
couples who have been unable to cryopreserve their
embryos for financial constraints?
Cryopreserved ‘spare’ embryos: As mentioned earlier,
it is a common practice to cryopreserve the spare
embryos following IVF as the cumulative pregnancy
rates improve following fresh and cryopreserved
embryo transferred after IVF. When a couple
achieves pregnancy either with the transfer of fresh/
cryopreserved embryos, all the cryopeserved embryos
may not be required by the couple. The first human
embryonic stem cell line was derived from a frozenthawed embryo1. This remains one of the main sources
of human embryos for derivation of hESC lines.
However, according to the ICMR Guidelines on ART6
(2012), “Consent shall need to be taken from the couple
for the use of their stored embryos by other couples or
for research, in the event of their embryos not being
used by themselves. This consent will not be required
if the couple defaults in payment of maintenance
charges after two reminders sent by registered post”.
That being the case, the question that arises is: can
cryopreserved embryos from couples who default in
making their payment of maintenance charges be used
for the derivation of ESC lines even in the absence of
a written consent?
The pro-life ethical concerns still dominate the
use of such embryos. And, the earlier worry about
intentionally subjecting women to over-stimulation
so as to have ‘spare’ cryopreserved embryos still
remains.
‘Poor’ quality discarded embryos: Embryos that are
discarded during the IVF procedure because of poor
morphology and a low likelihood for generating viable
pregnancies or surviving the cryopreservation process
are also a viable source of hES cells. It has been reported
that poor quality day three embryos judged on the basis
of morphological assessment have formed blastocysts
and give rise to hES cell lines7. However, early-arrested
or highly fragmented embryos only rarely yield cell
lines, whereas those that have achieved blastocyst
stage are a robust source of normal hES cells8.
Arrested human embryos do not resume normal
development during extended culture, yet most of
them contain a substantial number of living cells on
embryonic day six. Gavrilov et al9 reported that 72 per
cent of such embryos have <1 viable cell, 47 per cent
have <5 viable cells. They suggested that this class of
non-viable embryos could be a rich source of viable
cells for derivation of hESC lines9.
Attempts are also being made to develop strategies
for harvesting of live hESC from dead embryos10. the
inner cell mass (ICM) could be produced in 13.9 per
cent of poor quality embryos cultured11. Of the goodquality ICM, 15.4 per cent of those used in hESC
derivation attempts resulted in a novel line11.
Modifying culture medium to obtain cell lines from
poor quality embryos
Culturing poor-quality embryos in modified medium
containing human recombinant leukaemia inhibitory
factor and human basic fibroblast growth factor resulted
in a two-fold increase in the blastocyst formation rate
and a seven-fold increase over the derivation efficiency
in conventional medium12. All cell lines shared typical
human pluripotent stem cell features including similar
morphology, normal karyotypes, expression of alkaline
phosphatase, pluripotency genes, the ability to form
teratomas in SCID mice, and the ability to differentiate
into cells of three embryonic germ layers in vitro.
These data suggest that poor-quality embryos that have
reached the blastocyst stage in this modified culture
medium are a robust source for normal HESC line
derivation12.
Two new sibling human embryonic stem cell
lines BJNhem19 and BJNhem20, have been derived
and characterized from discarded grade III embryos
of Indian origin. The cells from these two sibling cell
lines showed normal diploid karyotype and continued
to express all pluripotency markers after long-term
continuous culture for over two years and passaged
over 200 times. However, BJNhem19 was unable to
generate teratomas (a unique characteristic of ESCs)
in nude or SCID mice or differentiate into beating
cardiomyocytes while the cardiac differentiation
capacity of BJNhem20 was greatly increased, and it
could generate beating cardiomyocytes13.
These reports indicate that some poor quality
embryos may develop into good ESCs but others may
Mehta: Sourcing human embryos for embryonic stem cell lines
not. But, these embryos remain a good potential, ethical
source for embryonic stem cell derivation.
Abnormally fertilized oocytes and aneuploid
embryos
Abnormally fertilized human oocytes depicted by
the presence of more than two pronuclei can develop
into normal appearing embryos. Such embryos are
discarded because of aneuploidy. Therefore, their use,
for generating ESC lines, would not conflict with the
interests of the donor as these in any case have to be
discarded. Many ART clinics offer embryo biopsy
for the removal of a single blastomere for genetic
diagnosis or screening for chromosomal anomalies.
Aneuploid embryos are not transferred and discarded.
Initially, human ESC lines were generated from
aneuploid embryos as these created a unique repository
of cell lines. The spectrum of aneuploidies in these
ESC lines reflects the range of common embryonic
chromosomal aberrations. Such aneuploid human ESC
lines represent an excellent model to study human
chromosomal abnormalities especially in the early
stages of development14.
Recent studies have demonstrated that genetically
normal ESC lines have been derived from aneuploid
embryos. Eleven stem cell lines were obtained from
41 embryos in 36 cultures15. The resulting stem cell
lines were karyotyped, and surprisingly, six of the nine
lines from aneuploid embryos were karyotypically
normal. Three lines from pre-implantation genetic
diagnosis (PGD) embryos were aneuploid exhibiting
trisomy 5, trisomy 16, and an iso chromosome 13,
respectively. None of the aneuploid lines presented
the same anomaly as the original PGD analysis. This
study demonstrated that normal stem cell lines could
be derived from biopsied developmentally abnormal
embryos offering specialty stem cell lines for research
into the clinically important aneuploidies. Mosacism
in human embryos is well known and this study
further demonstrates the emerging dominance of
the stem cell line by karyotypically normal cell15. In
another study, karyotype analysis of ESC lines derived
from aneurploid embryos showed a diploid female
karyotype16. Harkness et al4 also managed to derive
seven normal euploid ESC lines from 74 fresh PGS
screened aneuploid embryos. Thus, aneuploid embryos
detected after preimplantation genetic screening can
lead to the development of genetically normal ESC
lines.
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From unfertilized oocytes
One hESC line (RCM1) was obtained from a
failed-to-fertilize inseminated egg recovered by
parthenogenetic activation. Standard in vitro and in
vivo characterization revealed this line to possess all
of the properties attributed to a normal euploid hESC
line. Whole-genome single-nucleotide polymorphism
analysis further revealed that the line was biparental,
indicating that sperm penetration had occurred,
although parthenogenetic stimulation was required for
activation17.
Metaphase II stage oocytes have been
parthenogenetically activated or reinseminated with
donor sperm, then allowed to develop up to the
blastocyst stage. Fertilization occurred in 65 per cent
of the activated or reinseminated oocytes, which
resulted in a blastocyst formation rate of 8 per cent.
Evaluation of a number of developmentally important
genes in those embryos exhibiting normal development
revealed profile and levels of expression similar to
control embryos18. The use of artificial parthenogenesis
represents an alternative ethical source for pluripotent
cell lines19. Pluripotent stem cells (PSCs) derived
from parthenogenetically activated human oocytes
demonstrate the typical characteristics displayed
by human embryonic stem cells (hESCs) including
infinite division and in vitro and in vivo differentiation
into cells of all germ lineages20.
The use of unfertilized oocytes does not involve
the destruction of or harm to human embryos5 and,
therefore, does not face the ethical dilemma associated
with the use of embryos. As these technologies are
far from therapeutic, concerns over the morality of
embryonic stem cell derivation should not hinder their
advancement21.
From single blastomeres
Human embryonic stem cell lines have been
derived from biopsied single blastomeres. Chung and
colleagues22 succeeded in deriving mouse embryonic
stem cells from single blastomeres separated from
eight-cell-stage mouse embryos. Since this technique
sought to preserve the ability of the embryo to implant
and develop to birth, it theoretically could allow for the
banking of autologous hES cell lines for children born
from biopsied ex-corporeal embryos. Blastomeres are
removed from morula stage embryos and cultured until
multicell aggregates are formed. These blastomere-
110 INDIAN J MED RES, november (Suppl.) 2014
derived cell aggregates are plated into microdrops
seeded with mitotically inactivated feeder cells, and
then connected with neighbouring microdrops seeded
with green fluorescent protein-positive hES cells. The
resulting blastomere-derived outgrowths are cultured
in the same manner as blastocyst-derived hES cells23.
Chung et al24 have demonstrated that a single
blastomere can be removed to generate a human
embryonic stem cell line without prejudicing the
development of the biopsied embryo. Their method
stimulates new ideas about hESC formation, but ethicopolitical concerns remain.
Autologous nuclear transfer
Markoulaki et al25 have developed in mice a
variation of somatic cell nuclear transfer (SCNT), a
technique whereby the DNA of an unfertilized egg is
replaced by the DNA of a somatic cell, by blocking
the action of a gene [caudal type homeobox 2 (Cdx2)]
that enables the developing embryo to implant into the
uterus. By introducing this genetic defect in mouse
somatic cells prior to nuclear transfer, they created
cloned mouse embryos that generated pluripotent stem
cells just before arresting developmentally.
Another concept called altered nuclear transfer
(ANT) was proposed by William Hurlbut. Research
with mouse embryos carrying a mutation in the Cdx2
gene showed that these embryos failed to form a
trophectoderm and thus died at the blastocyst stage, but
not before giving rise to mouse embryonic stem cells.
Extrapolating from this mouse study, Hurlbut reasoned
that a Cdx2 genetic mutation introduced into a human
somatic cell prior to nuclear transfer might produce a
blastocyst that could produce human pluripotent stem
cells but lacked the biologic potential to develop into
a complete human being. He also suggested that these
possible ANT products should be viewed as complex
tissue cultures (i.e., bioengineered embryo-like
artifacts) rather than viable human embryos because
of their limited cellular systems26. Unfortunately, there
were many uncertainties surrounding ANT as a possible
source for human pluripotent stem cells. Meissner and
Jaenisch27 pointed out that it was unknown whether
Cdx2-deficient human embryos would behave just
like their mouse embryo counterparts, yielding
pluripotent human stem cells just before arresting at
the late blastocyst stage. To determine whether ANT
was feasible, efficient, and effective for research
and clinical applications in humans would require
significant amounts of time-consuming research and a
considerable diversion of resources that could be used
toward known methods for deriving hES cells.
Conclusions
Embryonic stem cells have great potential
application in transplantation and regenerative
medicine as well as improving our understanding of
developmental biology. There is a need for regularly
sourcing human embryos to develop a genetic bank
of embryonic stem cells. With an estimation of nearly
85000 ART cycles being done in India every year, it
should be possible to source human embryos either by
the conventional or novel means which can be used
for derivation of embryonic stem cells lines without
compromising on any of the ethical concerns.
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Reprint requests:Dr Rajvi H. Mehta, 111-115, Marathon Max, Hedgewar Chowk, L.B.S. Marg,
Mulund (West), Mumbai 400 080, Maharashtra, India
e-mail: [email protected]